Cyclohexanol, 4-(1,1-dimethylethyl)-, trans-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-14 to 2012-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2010-10-20

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

yes

Details on sampling:

Each test item concentration and the control were analytically verified at the beginning and the end of the test via GC-MS analysis. At test start samples were taken out of additional replicates provided without algae. At the end of the test samples were taken from additional replicates with algae and incubated under test conditions.

Vehicle:

no

Details on test solutions:

PREPARATION
- Stock solution: 100 mg/L, freshly prepared with dilution water
- Dispersion treatment: The test item was completely melted at ca. 70 - 75 °C before weighing out. The stock solution was stirred for 24 hours (1100 rpm) with a magnetic stirrer. Thereafter, the stock solution was treated for 20 min with ultrasound and after a separation phase of 1 hour (settlement of solid particles) the stock solution was taken from the homogeneous liquid phase.

APPLICATION
Application was carried out by adding appropriate volumes of the stock solution to the test concentrations.

CONTROL
Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Chlorophyta, Chlorophyceae
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen
- Age of inoculum (at test initiation): A four day old exponential growing preculture was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE•m-2•s-1 for 24 hours per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no data

Hardness:

no data

Test temperature:

room temperature (measured): min.: 22.5 °C, max.: 23.5 °C, mean value: 23 °C

pH:

measured values: between pH 7.99 - 8.10 (at start of the test), between 8.17 - 9.26 (at 72 hr)

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Test concentrations nominal: 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L (factor √10)
Measured concentrations: Measured concentrations at test start were in the range of 97 to 104% of the nominal values. Recovery rates at the end of the test were in the range of 100 to 106% of the nominal values. Thus, all effect values are based on nominal concentrations of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks, volume: 119 mL, with aluminium tops with PTFE seals.
- Test volume: 119 mL
- Initial cell density: Nominal: approximately 5 x 103 - 104 cells/mL; Current: 5466 cells/mL
- Control end cells density: 561652
- Incubation: The flasks were positioned randomly and repositioned daily.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Light intensity: 60 - 120 µE • m-2 • s-1
- Light regime: 24 hours/day light

GROWTH MEDIUM
- Nutrient medium Z according to LÜTTGE et al. (1994).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the guideline (OECD 201 medium)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Cell density was measured via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. The cell density was measured at the beginning of the test (after application) and every 24 hours. Dilution water was used as background signal.
- The pH-value at the beginning of the test was measured out of one additional replicate of each test concentration and control. At the end it was measured from pooled replicates per concentration level and control.
- The room temperature was measured continuously by a hygrothermograph.
- Light intensity was measured prior to test start.
- Microscopic evaluation of the cells at the start and at the end of the incubation was determined. Cells were checked for any unusual cell shapes, colour differences, chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of alga cells.

TEST CONCENTRATIONS
- Test concentrations nominal: 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L (factor √10)
- Range finding study: A non GLP preliminary range finding test was conducted under static conditions with four nominal test item concentrations ranging from 0.0100 to 100 mg/L set up in a geometric series with a dilution factor of 10.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence intervals: < 1.00 – 3.56

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence intervals: 13.6 – 18.9

Details on results:

CONCENTRATIONS
The test item concentrations were analysed at all concentration levels at test start and test end via GC-MS analysis. The measured concentrations at test start were in the range of 97 to 104 % of the nominal values. Recovery rates at the end of the test were in the range of 100 to 106 % of the nominal values. All effect values are given based on the nominal concentrations of the test item.

BIOLOGICAL DATA
Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities. All effect values are given based on nominal concentrations of the test item.

SELF FLUORESCENCE
No self fluorescence was found at the beginning of the definitive test at the nominal concentration level of 100 mg/L.

VALIDITY CRITERIA
- The cell growth increased 103-fold after 72 hours in the control (required: at least 16 fold).
- The temperature during the test was in the range of 21-24 °C.
- The difference from the mean was within ± 2 °C: min.: 22.5 °C, max.: 23.5 °C, mean value: 23.0 °C.
- The pH-value in the control replicates increased not higher than 1.5 units: from 8.05 to 9.26.
- The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures was 33.1 % (required: < 35 %).
- Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 6.17 % (required: < 7 %).

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA, batch number 1450864V, purity 100.3%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2012-04-10 to 2012-04-13. The reference item toxicity is in the valid range following test facility SOPS.

EC50 Values of the Reference Item based on nominal concentrations in mg/L (0-72 hours):

- Rate-related inhibition:
ErC50 = 0.718 (95 % confidence interval 0.699 - 0.737)
Valid Range (average ± 3 x SD) 0.845 ± 0.418

- Yield inhibition:
EyC50 = 0.422 (95 % confidence interval 0.386 - 0.466)
Valid Range (average ± 3 x SD) 0.452 ± 0.250

(SD = Standard deviation)

Reported statistics and error estimates:

EC-values and statistical analyses:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals of EC10-, EC20- and EC50-values were carried from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

NOEC and LOEC values:
NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is α=0.05. Normality Test failed for calculation of yield data. The highest concentration level with 100 % inhibition was excluded to pass Normality Test. Detection of not statistically significant differences were considered positive when inhibition values were > 10 %. These findings were marked “biologically significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant.

SOFTWARE:
The data for the tables in this report were computer generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures. Calculations were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		0.00	100		0	100
	2		0.00	100		0	100
	3		0.00	100		0	100
	Mean	(+)	0.00	100	#	0	100
32.0	1		0.463	69.8		16479	97.0
	2		0.376	75.5		11428	98.0
	3		0.483	68.5		17824	96.8
	Mean	(+)	0.441	71.3	(+)	15244	97.3
10.0	1		0.937	38.9		85459	84.6
	2		0.948	38.2		88532	84.1
	3		0.907	40.8		77681	86.0
	Mean	(+)	0.931	39.3	(+)	83891	84.9
3.20	1		1.40	8.52		362182	34.9
	2		1.47	3.86		450092	19.1
	3		1.40	8.52		362070	34.9
	Mean	(-)	1.43	6.97	(-)^1	391448	29.6
1.00	1		1.40	8.74		358420	35.6
	2		1.40	8.75		358308	35.6
	3		1.34	12.6		298596	46.3
	Mean	(+)	1.38	10.0	(+)	338441	39.2
Control	1		1.64			750252
	2		1.58			622299
	3		1.38			339795
	4		1.48			456722
	5		1.60			655861
	6		1.52			512188
	Mean		1.53			556186

#) = not included in NOEC calculation

1) = biologically significant (inhibition > 10 %)

Concentrations and Percent of the nominal concentration of the Test Item

Sampling date	2012-05-15 0 h (Fresh medium)		2012-05-18 72 h (Old medium)
Start of analysis	2012-05-15		2012-05-18
Nominal test item concentration [mg/L]	Test item
	Meas. conc.   [mg/L]	%	Meas. conc.   [mg/L]	%
100	104	104	103	103
32.0	32.2	101	33.8	106
10.0	9.72	97	10.2^1)	102
3.20	3.29	103	3.32^1)	104
1.00	1.02	102	1.00	100
Control	<LOQM		<LOQM

Meas. conc.   =Measured concentration (dilution factor taken into account)

%                     = Percent of the nominal concentration of the test item

LOQ_M              = Limit of quantification of theanalytical method(0.1mg test item/L)

1)                     = Mean value of two replicates (dilution factor taken into account), reanalysed on  2012-05-22.

METHOD VALIDATION

Linearity

The analytical system gave linear response in the range of nominal 5 to 75 µg/L of the test item. Linearity (r²) of the calibration curve was > 0.992.

Limit of Detection (LOD)

The limit of determination (LOD) of theanalytical methodwas determined to be 1 µg test item/L with asignal to noise value of 12.

Limit of Quantification (LOQ_M)

The limit of quantification (LOQ_M) of theanalytical methodwas fixed at 0.1 mg test item/L. The 1 x LOQ_Mand 100 x LOQ_Mwere checked by means of accuracy.

Accuracy, Precision and Specificity

Mean recoveries of 91 %x LOQ_Mand 91 % at 100 x LOQ_Mwere obtained indicating accuracy of the analytical method. The corresponding coefficients of variation of 8.10 % and 6.74 % indicated a sufficient precision of the analytical method. Specificity was confirmed because responses of blank samples were < 30 % of the 1 x LOQ_M.

Validity criteria fulfilled:

yes

Remarks:

see "Details on results"

Conclusions:

In this study the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (nominal concentrations): The EC-value with 95 % confidence intervals for inhibition of specific growth rate (ErC50) after 72 hours was 16.0 (13.6 – 18.9) mg test item/L. The ErC10 (72h) was 2.19 mg/L (95 %-CL: < 1.00 - 3.56 mg/L). The NOEC-value for the inhibition of specific growth rate was 3.20 mg test item/L. All effect values are given based on nominal concentrations of the test substance.

Description of key information

Key data: EC50(72 h) = 16.0 mg/L (growth rate); EC10(72h) = 2.19 mg/L (growth rate); NOEC(72 h) = 3.20 mg/L  (growth rate); LOEC(72 h) = 10.0 mg/L (growth rate)
(All effect levels are given based on nominal test item concentrations).

Key value for chemical safety assessment

EC50 for freshwater algae:

16 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L

Additional information

The toxicity test to aquatic algae performed according to EU Method C.3., OECD 201 and GLP was rated with RL=1 (reliable without restrictions) and used as a key study.

The results indicate that the test item inhibits the growth of the freshwater green alga Pseudokirchneriella subcapitatus after 72 hours with the following effect values (nominal test item concentrations) (measured concentrations within 80 - 120% of nominal values):

The EC₅₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rC₅₀) and yield (E_yC₅₀) after 72 hours were 16.0 (13.6 – 18.9) mg test item/L and 3.31 (1.80 – 5.75) mg test item/L, respectively.

The NOEC-values for the inhibition of specific growth rate and yield after 72 hours were 3.20 and < 1.00 mg test item/L, respectively.